Attenuated rna virus and applications thereof

ABSTRACT

The invention encompasses an attenuated RNA virus and methods of using an attenuated RNA virus. The RNA virus comprises, in part, an ion channel protein comprising a peptide tag.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional application No.60/973,870, filed Sep. 20, 2007, which is hereby incorporated byreference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under grant numberA1053629 and A1061252 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention encompasses an attenuated RNA virus, immunogeniccompositions comprising an attenuated RNA virus, and methods of using anattenuated RNA virus.

BACKGROUND OF THE INVENTION

RNA viruses include viruses that cause HIV, the common cold, hepatitis,influenza, polio, mumps, measles, SARS, Ebola, and other diseases.Vaccination against RNA viruses is one way of protecting populations,both human and animal, from these diseases. In particular, vaccinatingagainst influenza A virus is one means of controlling morbidity andmortality resulting from annual influenza epidemics.

Generally speaking, the immune response induced by a live, attenuatedvaccine is believed to be superior to that induced by inactivated virus,resulting in increased protection against antigenic-drift variants andother antigenic subtypes. Live attenuated vaccines, however, potentiallypose a greater risk to the host than an inactivated virus. In addition,methods used to attenuate the virus may affect the ability of the virusto induce a protective immune response in the host. Consequently, thereis a need in the art for safe live attenuated vaccines that induce aprotective immune response against an RNA virus in a host.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses an isolated RNA virus.The virus comprises an ion channel protein comprising a peptide tag.

Another aspect of the invention encompasses an immunogenic composition.The composition comprises at least one live attenuated RNA virus, thevirus comprising an ion channel protein comprising a peptide tag.

Yet another aspect of the invention encompasses a method for inducing aprotective immune response in a subject. The method comprisesadministering to the subject an immunogenic composition comprising atleast one live attenuated RNA virus, the virus comprising an ion channelprotein comprising a peptide tag.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the in vitro replication of recombinant influenza Aviruses in MDCK cells. (A) presents a graph of the mean TCID₅₀ valuesand standard errors for the indicated virus at several time points. (B)is a plot of the diameters of 25 plaques for the indicated viruses. Thesolid horizontal line indicates the average plaque diameter.

FIG. 2 illustrates the in vivo replication and pathogenesis ofrecombinant influenza A viruses. Mice (n indicates the number of mice)were inoculated intranasally with the indicated viruses at the indicateddoses. (A) presents a plot of survival over the course of two weeks. (B)B is a plot of body weight over the two weeks. Mouse weight wasnormalized to the weight at the time of infection and the average andstandard error are graphed. At the indicated days post infection, viralloads were determined by TCID₅₀ in tracheas (C) and lungs (D). Eachsolid, horizontal line represents the average. The dashed, horizontalline indicates the limit of detection.

FIG. 3 illustrates that infection with rWSN M2myc protects mice from alethal challenge with rWSN. Mice previously infected with rWSN M2mycwere challenged with a lethal dose of rWSN and monitored for mortality(A) and weight loss (B). Mouse weight was normalized to the weight atthe time of infection and the average and standard error are graphed.Serum was collected from mice that survived infection with either rWSNor rWSN M2myc and from immunized mice that survived a subsequentchallenge with rWSN. (C) presents a plot of TCID₅₀ values for eachcondition.

FIG. 4 illustrates a graph showing that immunization with rWSN M2myc(H1N1) viruses induces immunity to an antigenically distinct influenzavirus, A/HK/68 (H3N2).

DETAILED DESCRIPTION OF THE INVENTION

In an attempt to differentially tag a virus, an antibody epitope tag wasattached to the carboxyl terminal end of an ion channel protein of thevirus. As expected, the addition of this tag did not alter the functionof the ion channel protein or the in vitro replication of the virus.Unexpectedly, however, the addition of the tag attenuated the virulenceof the virus, as judged by its inability to cause disease as compared tothe parental strain of the virus. Furthermore, it was discovered thatthe attenuated virus comprising the peptide-tagged ion channel was ableto provide protective immunity, indicating that it may be used as animmunogenic composition. This invention, therefore, encompasses theattenuated virus comprising a peptide-tagged ion channel protein,immunogenic compositions comprising the attenuated virus, and methods ofusing the compositions comprising the attenuated virus to provideprotective immunity.

I. Isolated RNA Virus

One aspect of the present invention provides an isolated RNA viruscomprising an ion channel protein comprising a peptide tag. Although thefunction of the peptide-tagged protein is not altered, the ability ofthe isolated RNA virus of the invention to induce disease is attenuatedrelative to that of a virulent strain of the same virus. Thus, theisolated RNA virus of the invention may be used to provide protectiveimmunity.

(a) Types of Viruses

The parental strain of the isolated virus of the invention may be a wildtype, a recombinant, or an attenuated virus. The attenuated virus mayhave at least one mutation that confers cold adaptation or alteredgrowth, replication, and/or virulence. In general, the virus will be anRNA virus. For example, it may be a positive-sense single strand RNAvirus, a negative-strand RNA virus, or a retrovirus. Non-limitingexamples of suitable positive-strand RNA viruses include coronavirus,Dengue fever virus, hepatitis A, C, and E viruses, poliovirus,rhinovirus, Ross River virus, rubella virus, Sindbis virus, West Nilevirus, and yellow fever virus. Non-limiting examples of suitablenegative-strand RNA viruses include Ebola virus, hantavirus, humanparainfluenza virus types 1-4, influenza A, B, and C viruses, Lassavirus, Marburg virus, measles virus, mumps virus, Nipah virus,respiratory syncytial virus, rabies virus, and rinderpest virus.Suitable examples of retroviruses include human immumodeficiency virus,human T-lymphotropic virus, and feline immumodeficiency virus. Exemplaryviruses include influenza A, B, and C viruses, coronavirus, hepatitis Cvirus, and human immumodeficiency virus. An especially preferred virusis an influenza A virus. Influenza A virus may be further subdividedinto subtypes on the basis of the two main surface glycoproteinshemagglutinin (HA) and neuraminidase (NA). There are sixteen knownvariations of the HA protein (H1-H15) and nine known variations of theNA protein (N1-N9). Especially preferred influenza A subtypes includeH1N1, H1N2, H3N2, and H5N1.

(b) Ion Channel Protein Comprising a Peptide Tag

A number of RNA viruses encode small integral membrane proteins thathave ion channel activity. The ion channel protein may facilitate entryof the virus into a susceptible cell by functioning as a proton channelthat allows for acidification of the interior of the viral particle.Furthermore, the ion channel protein may play a role in infectious viralparticle assembly. Non-limiting examples of suitable ion channelproteins include the M2 protein of the influenza A virus, the BM2protein of the influenza B virus, the CM2 protein of the influenza Cvirus, the E protein of coronaviruses, the p7 protein of the hepatitis Cvirus, and the Vpu and/or Vpr proteins of the human immunodeficiencyvirus.

The ion channel protein of the isolated RNA virus of the invention has apeptide tag. The peptide tag may range from about 3 amino acids to about30 amino acids in length. In one embodiment, the peptide tag may rangefrom about 3 amino acids to about 10 amino acids in length. In anotherembodiment, the peptide tag may range from about 10 amino acids to about15 amino acids in length. In an alternate embodiment, the peptide tagmay range from about 15 amino acids to about 20 amino acids in length.In still another embodiment, the peptide tag may range from about 20amino acids to about 25 amino acids in length. In yet anotherembodiment, the peptide tag may range from about 25 amino acids to about30 amino acids in length.

In one embodiment, the peptide tag may comprise a random amino acidsequence. In another embodiment, the peptide tag may comprise a knownamino acid sequence. The known amino acid sequence may have antibodiesgenerated against it, or there may be no known antibodies generatedagainst the sequence. In yet another embodiment, the peptide tag may bean antibody epitope tag for which commercial antibodies are available.Non-limiting examples of suitable antibody epitope tags are listed inTable A. An exemplary antibody epitope tag is the myc antibody epitopetag.

TABLE A Antibody Epitope Tags Sequence Tag Name (N to C termini) SEQ IDNO: myc EQKLISEEDL  1 AcV5 SWKDASGWS  2 AU1 DTYRYI  3 AU5 TDFYLK  4 EGAPVPYPDPLEPR  5 ECS DDDDK  6 E2 SSTSSDFRDR  7 FLAG DYKDDDDK  8 Glu-GluEYMPME  9 HSV QPELAPEDPEDC 10 KT3 KPPTPPPEPET 11 S KETAAAKFERQHMDS 12 S1NANNPDWDF 13 T7 MASMTGGQQMG 14 V5 GKPIPNPLLGLDST 15 VSV-G YTDIEMNRLGK 166xHis HHHHHH 17

The location of the peptide tag in the ion channel protein can and willvary. In one embodiment, the peptide tag may be located at the aminoterminal end of the ion channel protein. In another embodiment, thepeptide tag may be located internally in the ion channel protein. Forexample, the peptide tag may be located between the amino terminus andthe transmembrane domain of the ion channel protein. Alternatively, thepeptide tag may be located between the carboxyl terminus and thetransmembrane domain of the ion channel protein. In a preferredembodiment, the peptide tag may be located at the carboxyl terminal endof the ion channel protein.

The ion channel protein may be tagged with a peptide using molecularbiology procedures well known to those of skill in the art (Sambrook etal. (1989) “Molecular Cloning: A Laboratory Manual,” 2^(nd) Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). For example,the nucleotide sequence encoding the peptide tag may be introduced atthe 5′ end, the 3′ end (e.g., in place of the stop codon), or within thenucleotide sequence of the ion channel protein. Techniques for isolatingviral RNA and transcribing it into cDNA are also well known in the art(e.g., Hoffman et al. (2000) Proc. Natl. Acad. Sci. USA 97:6108-6113).The recombinant virus encoding the peptide-tagged ion channel proteinmay be recovered using standard methodologies. That is, positive-strandRNA viruses may be recovered using the techniques of Racaniello andBaltimore (Science 214:916-919, 1981) and/or Kaplan et al. (Proc. Natl.Acad. Sci. USA 82:8424-8428, 1985), both of which are incorporated byreference in their entirety. Negative-strand RNA viruses may berecovered by the reverse genetic approach of Neumann et al. (Proc. Natl.Acad. Sci. USA 96:9345-9350, 1999), which is hereby incorporated byreference in its entirety. The presence of the tagged nucleotidesequence may be verified using well-known procedures, such as PCR,reverse transcriptase PCR, and/or nucleotide sequencing. Similarly, thepresence of the peptide-tagged ion channel protein and its levels ofexpression may be determined using standard techniques, such as Westernblotting and/or ELISA assays.

In one embodiment, the isolated RNA virus may be a humanimmunodeficiency virus and the ion channel protein may be a Vpu proteinand/or Vpr protein. In another embodiment, the isolated RNA virus may bea hepatitis C virus and the ion channel protein may be a p7 protein. Instill another embodiment, the isolated RNA virus may be a coronavirusand the ion channel protein may be an E protein. In another alternateembodiment, the isolated RNA virus may be an influenza C virus and theion channel protein may be a CM2 protein. In yet another embodiment, theisolated RNA virus may be an influenza B virus and the ion channelprotein may be a BM2 protein. In a preferred embodiment, the isolatedRNA virus may be an influenza A virus and the ion channel protein may bean M2 protein. In another preferred embodiment, the isolated RNA virusmay be an influenza A virus, the ion channel protein may be an M2protein, and the peptide tag may be at the carboxyl terminus of theprotein. In an especially preferred embodiment, the isolated RNA virusmay be an influenza A virus, the ion channel protein may be an M2protein, and the carboxyl terminal peptide tag may be the myc antibodyepitope tag.

(c) Properties of the Isolated RNA Virus

In general, the function of the peptide-tagged ion channel protein issimilar to that of the native (untagged) ion channel protein. The ionchannel activity of the protein may be measured using techniques knownto those of skill in the art. For example, ion channel activity may bemeasured directly in mammalian cells (e.g., Chizhmakov et al. (1996) J.Physiol. 494(Pt. 2):329-336), indirectly in Xenopus oocytes (e.g.,Holsinger et al. (1994) J. Virol. 68:1551-1563), or indirectly via thefowl plague virus (FPV) HA protein rescue assay (e.g., McCown and Pekosz(2005) J. Virol. 79(6):2595-3605). Each of the afore mentioned articlesis incorporated by reference in its entirety.

The in vitro replication of the isolated RNA virus of the invention isgenerally similar to that of a wild type strain of the same species ofvirus. Viruses may be grown in a number of primary cells, includingmonkey kidney, calf kidney, hamster kidney and chicken kidney. Virusesmay also be grown in continuous cell lines, including Madin-Darby caninekidney (MDCK) cells, 293T human embryonic kidney cells, African greenmonkey kidney epithelial (Vero) cells, Madin-Darby bovine kidney (MDBK)cells, baby hamster kidney (BHK) cells, North African green monkeykidney fibroblast (CV-1) cells, Chinese hamster ovary (CHO) cells, TM4mouse Sertoli cells, HeLa human cervical epitheloid carcinoma cells,buffalo rat liver (BRL) cells, WI-38 human diploid lung cells, Hep G2human hepatocellular carcinoma cells, and mouse mammary tumor (MMT)cells. The optimal culture conditions (i.e., media, supplements,temperature, CO₂ concentration, etc.) for a particular type of cell, aswell as means to infect cells with the particular virus are known tothose skilled in the art.

Virus replication may be determined by a variety of techniques that areknown to those skilled in the art. For example, the viral titer may bedetermined using a tissue culture infectious dose₅₀ (TCID₅₀) assay or aplaque forming unit (pfu) assay. A TCID₅₀ unit is the amount of a virusthat results in a cytopathic effect in 50% of the infected cells. Virusreplication may also be monitored with a plaque assay in which thenumber and/or diameter of plaques are quantified and/or the morphologyof the plaques is denoted. Still another method that may be used tomonitor virus replication is a hemagglutination assay. Alternatively,virus replication may be determined by directly counting the number ofviral particles using an electron microscope. Additionally, virusreplication may also be monitored with a PCR assay, an antibody basedassay, or a branched DNA assay.

The in vivo replication of the isolated RNA virus of the invention isgenerally reduced relative to that of a wild type strain of the samespecies of RNA virus. In some embodiments, the in vivo replication isreduced by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 logs, or more, relative tothat of a wild type strain. A virus may be administered to a subject bya variety of means, including, but not limited to, transmucosally (e.g.orally, nasally, ocularly, or rectally), transdermally, parenterally(e.g., via intravenous injection, as well as, intra-arteriole,intramuscular, intradermal, subcutaneous, intraperitoneal,intraventricular, and intracranial), or topically. The replication ofthe virus in the subject may be determined by the same techniquesmentioned above for in vitro replication. Depending upon the type ofvirus, the viral load may be determined in a body fluid (e.g., blood,serum, saliva, cerebrospinal fluid, pleural fluid, lymphatic fluid,milk, sputum, semen, and urine), a cell, a cell extract, or a tissuesample. The body fluid, cells, or tissue sample may be obtained bybiopsy or surgery. Those of skill in the art will appreciate that thetype of body fluid or tissue analyzed will depend upon the particulartype of virus. For example, replication of influenza A virus may bemonitored in cells and/or fluid from the lungs or trachea.

Advantageously, the ability of the isolated RNA virus of the inventionto cause disease is attenuated relative to that of a wild type strain ofthe same virus. The isolated RNA virus of the invention, therefore, isan attenuated, less virulent virus. Preferably, viral virulence may bemonitored in an appropriate animal model of infection. The pathogenicityor virulence of a virus may be measured via a variety of means. Forexample, the morbidity of the viral disease may be assessed bymonitoring weight loss, changes in body temperature, fluid loss, and/orprotein or enzyme levels. The mortality of the viral disease may beassessed by monitoring survival and/or death. Those of skill in the artwill know the appropriate test or measurement to use to assess thevirulence of the particular virus.

Furthermore, the isolated RNA virus of the invention generally elicitsan immune response in the subject. The immune response may be a humoralresponse (i.e., mediated via B cells) and/or a cell mediated response(i.e., mediated via T cells). The cell mediated response may be acytotoxic T lymphocyte (CTL) and/or a delayed type hypersensitivity(DTH) response. Techniques to assess immune responses are well known inthe art (e.g., see Harlow and Lane (1988) “Antibodies. A LaboratoryManual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).In exemplary embodiments, the induced immune response generally protectsthe subject against challenge with a virulent stain of the same speciesof virus. Thus, the isolated RNA virus of the invention may be used toprotect a subject from disease caused by a virulent species, strain, orsub-type of the virus.

II. Immunogenic Composition

Another aspect of the present invention provides an immunogeniccomposition comprising at least one live attenuated RNA virus of theinvention, as described above in section I. Administration of animmunogenic composition of the invention to a subject typically does notinduce disease, but rather elicits a humoral and/or cellular immuneresponse and protects the subject from challenge against a virulentstrain of the same or a closely related virus.

To make an immunogenic composition, the attenuated RNA virus of theinvention will generally be produced in host cells that have beenapproved and certified according to the WHO requirements for vaccineproduction (Mizrahi, ed., Viral Vaccines, Wiley-Liss Inc., NY, pp. 3960, 1990). WHO certified, or certifiable, continuous cell lines arepreferred for producing the immunogenic composition. The requirementsfor certifying such cell lines include characterization with respect toat least one of genealogy, growth characteristics, immunologicalmarkers, virus susceptibility, tumorigenicity, and storage conditions,as well as by testing in animals, eggs, and cell culture. Suchcharacterization is used to confirm that the continuous cell lines arefree from detectable adventitious agents. In some countries, karyologymay also be required. In addition, tumorigenicity is preferably testedin cells that are at the same passage level as those used for vaccineproduction. Suitable host cells include embryonated hens' eggs, Verocells, or other mammalian cells. Non-limiting examples of cell linesthat may be suitable include, but are not limited to, mammalianfibroblast or epithelial cells maintained as continuous cell lines.Further non-limiting examples include Vero, MDCK, 293T, BK-21 and CV-1cells, readily available from commercial sources (e.g., ATCC, Rockville,Md.).

In some embodiments, an attenuated RNA virus of an immunogeniccomposition may have at least one additional attenuating mutation, inaddition to the peptide-tagged ion channel protein. The additionalmutation may be in a gene that affects cold adaptation, replication,growth control, or virulence. For example, in one embodiment, theattenuated RNA virus of an immunogenic composition may be an influenza Avirus comprising a peptide-tagged M2 protein, as well as a deletion inthe NS1 coding region. In another embodiment, the attenuated RNA virusof an immunogenic composition may be an influenza A virus comprising apeptide-tagged M2 protein, as well as an altered HA cleavage site. Instill another embodiment, the attenuated RNA virus of an immunogeniccomposition may be an influenza A virus comprising a peptide-tagged M2protein, as well as an altered temperature sensitivity.

In other embodiments, at least one gene that encodes an antigenic viralprotein of interest may be introduced into the attenuated RNA virus ofthe invention comprising an immunogenic composition. For example, genesencoding HA and/or NA proteins of influenza A may be introduced into anattenuated influenza A virus comprising a peptide tagged M2 protein.

In alternate embodiments, an immunogenic composition may furthercomprise at least one additional live attenuated virus (i.e., inaddition to the attenuated virus of the invention). The additionalattenuated virus may harbor temperature-sensitive mutations that limitits growth in the natural host. Furthermore, the additional attenuatedvirus may have mutations in genes that reduce its replication ormutations in virulence genes that reduce its virulence. In anotherembodiment, an immunogenic composition may further comprise at least oneinactivated virus. The inactivated virus may be killed by heat orchemical (formaldehyde) treatment. An inactivated virus is, therefore,unable to replicate, but maintains its antigenic constitution andimmunogenicity. In yet another embodiment, an immunogenic compositionmay further comprise a purified viral protein, such as a viral coatprotein, a surface glycoprotein, etc.

In a preferred embodiment, an immunogenic composition may comprise atleast one live attenuated influenza A virus carrying a carboxyl terminaltagged M2 protein. The influenza A virus may be an H1N1 subtype, an H1N2subtype, an H3N2 subtype, an H5N1 subtype, or a combination thereof. Inanother preferred embodiment, an immunogenic composition may comprise anH1N1 subtype of an influenza A virus, an H3N2 subtype of an influenza Avirus, and an influenza B virus. In still another preferred embodiment,an immunogenic composition may comprise an influenza A virus and aninfluenza B virus.

An immunogenic composition of the invention may further comprise one ormore adjuvants, carriers, and/or excipients. Adjuvants are typicallysubstances that generally enhance the immune response of a subject to aspecific antigen. Suitable adjuvants include, but are not limited to,complete Freund's adjuvant, incomplete Freund's adjuvant, otherbacterial cell wall components, aluminum-based salts, calcium-basedsalts, silica, polynucleotides, toxoids, serum proteins, viral coatproteins, other bacterial-derived preparations, gamma interferon, blockcopolymer adjuvants, such as Hunter's Titermax adjuvant (available fromCytRx Corp. Inc., Norcross, Ga.), Ribi adjuvants (available from RibiImmunoChem Research, Inc., Hamilton, Mo.), and saponins and theirderivatives, such as Quil A (available from Superfos Biosector A/S,Denmark). Carriers are typically compounds that increase the half-lifeof a therapeutic composition in a treated subject. Suitable carriersinclude, but are not limited to, polymeric controlled releaseformulations, biodegradable implants, liposomes, oils, esters, andglycols. An excipient is generally an inert substance used as a diluentor a vehicle for delivery. Examples of suitable excipients includewater, saline, Ringer's solution, dextrose solution, Hank's solution,and other aqueous physiologically balanced salt solutions. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientsmay also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer, and Tris buffer, while examples ofpreservatives include thimerosal, m- or o-cresol, formalin and benzylalcohol. Preferably, the adjuvant, carrier, and/or excipient arepharmaceutically acceptable.

The term “pharmaceutically acceptable” refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human. Preferably, asused herein, the term “pharmaceutically acceptable” means approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans.

III. Method for Providing Protective Immunity

A further aspect of the invention encompasses a method for providingprotective immunity to a subject by administering an immunogeniccomposition comprising at least one live attenuated RNA virus comprisinga peptide tagged ion channel protein. Immunogenic compositions weredescribed above in section II and the attenuated RNA virus was describedabove in section I.

An immunogenic composition of the invention may be administered to asubject by a variety of means. Suitable means include, but are notlimited to, transmucosal administration (e.g., oral, intranasal,intraocular, or rectal), transdermal administration, parenteraladministration (e.g., via intravenous injection, as well as,intra-arteriole, intramuscular, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial injection), ortopical administration. For example, in embodiments in which theattenuated virus of an immunogenic composition is an influenza virus, apreferred method to administer the immunogenic composition is byintranasal administration. Such administration may be accomplished byuse of a syringe fitted with cannula, or by use of a nebulizer fittedover the nose and mouth of the subject.

The dose of an immunogenic composition administered to a subject can andwill vary, depending upon a variety of factors. In general, animmunogenic composition will comprise an amount of live attenuated RNAvirus of the invention that is sufficient to protect the subject, for asuitable period of time, from challenge with a virulent strain of thevirus. The amount of live attenuated RNA virus may range from about 10³TCID₅₀ units of virus to about 10⁸ TCID₅₀ units of virus, or morepreferably from about 10⁵ TCID₅₀ units of virus to about 10⁷ TCID₅₀units of virus.

The number of doses and/or frequency of dose administration can and willvary. In one embodiment, the subject may be protected from challengewith a virulent species of the virus by administering a single dose ofan immunogenic composition of the invention. A suitable single dose is adose that is capable of protecting the subject from challenge with avirulent species of the virus when administered one time. In anotherembodiment, multiple doses may be administered over a period of timethat ranges from about two weeks to about two years. In still anotherembodiment, a booster dose may be administered at some interval of timeafter the original administration. Booster administrations preferablyare administered when the immune response of the subject becomesinsufficient to protect the subject from from challenge with a virulentstrain of the virus.

The efficacy of an immunogenic composition of the invention to generatean immune response may be tested in a variety of ways. Humoral immunitymay be assessed by a variety of methods, including the detection ofantibodies via an immunoassay, such as an ELISA assay, a Western blot, adot blot, a radioimmune assay, a qualitative immunoassay, or acolorimetric assay, or a hemagglutination inhibition (HAI) test.Furthermore, if the immunogenic composition comprises an attenuated RNAvirus comprising an antibody epitope tagged ion channel protein, then animmunized subject may be differentiated from an infected subject. Animmunized subject may generate antibodies against the epitope tag, aswell as antibodies against viral proteins or other antigens.Additionally, cellular immunity may be monitored by the detection and/oranalysis of a population of activated T cells (e.g., via the detectionof specific cytokines synthesized and secreted by the activated cells).For example, a specific cytokine may be detected via an ELISA assay,fluorescent activated flow cytometry, or an immunofluorescent microscopyassay.

An immunogenic composition of the invention may be administered to avariety of subjects. In general, the subject will be a human or ananimal that is susceptible to the disease caused by the RNA virus ofinterest. The subject may be a mammal, such as a human, a companionanimal, such as cat or a dog, an agricultural animal, such as dairycattle, beef cattle, a goat, a sheep, a swine, a horse, or anotherequid, a zoo animal, such as a primate, a lion, a mink, etc., alaboratory animal, such as a mouse, rat, or hamster, or a marine mammal,such as a whale or a seal. The subject may also be a domestic fowl, suchas a chicken, goose, or duck, a game fowl, or an aquatic bird. In anexemplary embodiment, the subject may be a human. While the age and thegeneral health status of the subject may vary, the subject is generallynot immunocompromised.

In general, administration of an immunogenic composition comprising anattenuated RNA virus comprising a peptide tagged ion channel proteininduces an immune response, provides protective immunity, and preventsdisease caused by a virulent strain of the virus.

As various changes could be made in the above compositions and methodswithout departing from the scope of the invention, it is intended thatall matter contained in the above description shall be interpreted asillustrative and not in a limiting sense.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below.

The term, “attenuated,” as used here, refers to a virus that is capableof stimulating an immune response and creating immunity but not causingillness.

As used herein, the term, “peptide tag,” refers to an exogenous aminoacid sequence that is added to a protein.

As used herein, the phrase “protective immune response,” or “protectiveimmunity,” refers to an immune response in a host that protects the hostfrom challenge with a virus of the same species. A host is “protectedfrom challenge” when the immune response contributes to the lessening ofany symptoms associated with infection of a host with the virus. Forexample, a protective immune response against influenza will induce animmune response that helps to ameliorate symptoms associated withinfluenza infection or reduces the morbidity and mortality associatedwith influenza infection. The use of the term “protective” in thisinvention does not necessarily require that the host is completelyprotected from the effects of the virus.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. Those of skill in the art should, however, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES

The following examples illustrate various embodiments of the invention.

Example 1 Epitope Tagging of the M2 Protein and in vitro VirusReplication

In order to further characterize the role of the M2 cytoplasmic tail inthe influenza A life cycle, a recombinant influenza A virus wasgenerated in which the myc epitope tag was attached to the carboxylterminus of the M2 protein. The addition of an antibody epitope tag tothe M2 protein cytoplasmic tail does not alter the ion channel activityor expression of the protein (Sakaguchi et al., (1997) Proc. Natl. Acad.Sci. USA 94:5000-5005).

The recombinant influenza A/WSN/33 virus that contained the myc-epitope(rWSN M2myc) was generated using standard procedures. Briefly, thenucleotide sequence (5′-GAGCAGAAGCTGATCTCCGAGGAAGACCTG-3′, SEQ ID NO:18)of the myc epitope tag (amino acids, EQKLISEEDL, SEQ ID NO:1) wasintroduced in place of the M2 stop codon in the vector pHH21 M segment.This plasmid encodes the M segment of the A/WSN/33 strain of influenza Avirus, see McCown and Pekosz (2005) and Neumann et al. (1999). Therecombinant virus encoding this sequence was recovered via standardmethodology, as described by McCown and Pekosz (2005) and Neumann et al.(1999). The presence of the myc epitope nucleotide sequences wasverified by reverse transcriptase polymerase chain reaction followed byDNA sequencing. The presence of the myc epitope in the M2 protein wasverified by Western blotting of virus-infected cell lysates withantibodies that recognize the M2 protein or the myc epitope.

The in vitro replication of this engineered virus was indistinguishablefrom that of the parental rWSN virus. Madin-Darby canine kidney cells(MDCK) were infected with these viruses at low MOI (FIG. 1A) or highMOI. After a 1 hr-incubation period, the inoculum was removed and thecells were washed with media and incubated at 37° C. At particular times(i.e., 12, 24, 38, and 72 hr) after infection, the infected cellsupernatant was sampled and infectious virus titers determined byTCID₅₀. The rWSN M2myc and the parental rWSN viruses were also analyzedby plaque assay on MDCK cells. The cells were harvested at 3 days postinfection and the plaque diameter of at least 25 plaques was measuredwith a micrometer. The plaque morphology and diameter of the viruses wasalso nearly identical between the viruses (FIG. 1B), suggesting theaddition of these amino acids to the M2 protein did not have an adverseeffect on virus replication.

Example 2 Virulence of M2 Epitope Tagged Viruses

To assess the virulence of rWSN M2myc, 6-week-old Balb/c mice wereanesthetized and inoculated intranasally with varying doses of rWSNM2myc or rWSN. The mice were weighed daily as a surrogate forvirus-induced morbidity, and mortality was also monitored for two weeks.The rWSN M2myc virus did not induce mortality in mice while thecorresponding dose of rWSN showed the expected amount of mortality (FIG.2A). Furthermore, mice inoculated with rWSN M2myc had significantlyreduced weight loss as compared to those inoculated with rWSN,indicating the recombinant virus had a drastically reduced virulence(FIG. 2B).

At day 3 and day 5 post infection, three mice from each group weresacrificed and the tracheas and lungs were removed. The viral load wasdetermined in each organ by TCID₅₀. Analysis of the viral load in thetracheas (FIG. 2C) and lungs (FIG. 2D) of virus-infected mice revealedthat the rWSN M2myc virus replicated to significantly lower titers thanthe parental rWSN virus. Taken together, these data indicate that theaddition of a myc-epitope to the carboxyl terminus of M2 had littleeffect on virus replication in vitro, but significantly attenuated virusreplication and virulence in the mouse model of infection.

Example 3 Attenuated Virulence is Due to Peptide Tag and not AdditionalNucleotide Sequences

In order to determine whether the addition of amino acids to the M2cytoplasmic tail or simply the presence of additional nucleotidesequences in the engineered virus was mediating the loss of virusvirulence, the authentic M2 stop codon was introduced into the M2myccoding sequence (rWSN M2stopmyc). This resulted in the production of anM2 protein that did not contain the myc epitope tag, but the viralgenome did contain the nucleotide sequence for the epitope tag. Thisvirus replicated in a manner indistinguishable from rWSN or rWSN M2 mycafter low MOI infection (FIG. 1A) and with respect to plaque size andmorphology (FIG. 1B), however, it regained the ability to causemortality in mice (FIG. 2A). These data indicate that the presence ofthe amino acids at the carboxyl terminus of the M2 protein, but not theadditional nucleotides present in the viral RNA, was responsible forattenuating virus virulence in vivo.

Example 4 M2 Epitope Tagged Viruses as Vaccines

Since the rWSN M2myc virus replicated but did not cause significantmorbidity or mortality in infected mice, its potential as a live,attenuated influenza vaccine was assessed. Mice that had been infectedwith rWSN M2myc were challenged 28 days post infection with a lethaldose of rWSN (i.e., 10⁶ pfu was administered intranasally). Irrespectiveof the virus dose, animals that had been previously infected with rWSNM2myc survived rWSN challenge (FIG. 3A) and had very little weight loss(FIG. 3B) as compared to age-matched, naïve mice. The protectionafforded by rWSN M2myc infection was equivalent to that seen in animalsthat were given a sublethal (10³ pfu) dose of rWSN (FIGS. 3A and 3B).Although it must be noted, that the morbidity induced by sublethal rWSNinfection was significantly greater than that observed with any dose ofrWSN M2myc (FIG. 2B).

Serum was collected from mice either prior to or 28 days after rWSNchallenge and the neutralizing antibody titers against rWSN determined.For this, dilutions of sera were incubated with 10³ pfu of rWSN, thenanalyzed in sextuplate by TCID₅₀. The serum concentration that inhibitedcytopathic effect in 50% of the wells was assessed after 4 days. Thelevel of neutralizing antibody titers in rWSN M2myc immunized mice werecomparable to those in mice immunized with a sublethal rWSN (FIG. 3C).Neutralizing antibody titers rose slightly after challenge. Takentogether, these data indicate that the addition of a carboxyl terminalepitope tag to the M2 protein resulted in significant attenuation ofvirus virulence but the immune response induced by virus infection wasable to provide protection against a lethal influenza A virus challenge.

Example 5 Protection Against Challenge with Different Surface Proteins

Mice were immunized with rWSN M2 myc, or viruses containing shorttruncations of the myc sequence. The myc epitope is 10 amino acids long,the viruses have this sequence truncated to 9, 8 or 6 amino acids inlength. These truncations maintain the attenuation of the virus. Theimmunized mice were then challenged with a virus that has differentsurface proteins from the rWSN M2myc viruses. This virus, A/HK/68 H3N2is lethal to unimmunized mice but does not kill mice immunized with therWSN M2myc virus, indicating that infection with the rWSN M2myc viruseshas induced an immunity that protects against different influenza Avirus strains. With the current influenza vaccines, separate componentsfor H1N1 and H3N2 viruses must be included to get immunity, as thesevaccines can't induce protection against antigenically unrelatedviruses. The current influenza vaccines induce specific antibodies thattarget the H1, H3, N1 or N2 proteins and these antibodies do notrecognize the other proteins. Infection with rWSN M2myc is inducing animmunity which allows for protection from antigenically unrelatedviruses. This means that one vaccine virus might be able to induceprotection against two different influenza A virus strains, which wouldbe a significant improvement over current influenza vaccines.

1. An isolated RNA virus, wherein the virus comprises an ion channelprotein comprising a peptide tag.
 2. The isolated RNA virus of claim 1,wherein the peptide tag is from about 3 amino acids to about 30 aminoacids in length.
 3. The isolated RNA virus of claim 1, wherein thepeptide tag is at the carboxyl terminus of the ion channel protein. 4.The isolated RNA influenza virus of claim 1, wherein replication of thevirus in vitro is similar to that of a virulent strain of the samevirus, and the replication and virulence of the virus in a host isreduced relative to that of a virulent strain of the same virus.
 5. Theisolated RNA virus of claim 1, wherein the virus is influenza A, the ionchannel protein is M2, and the peptide tag is at the carboxyl terminusof the protein.
 6. The isolated RNA virus of claim 5, wherein thepeptide tag is the myc antibody epitope tag, whose amino acid sequenceconsists of EQKLISEEDL (SEQ ID NO:1).
 7. An immunogenic composition,wherein the composition comprises at least one live attenuated RNAvirus, the virus comprising an ion channel protein comprising a peptidetag.
 8. The immunogenic composition of claim 7, wherein the peptide tagis from about 3 amino acids to about 30 amino acids in length.
 9. Theimmunogenic composition of claim 7, wherein the peptide tag is at thecarboxyl terminus of the ion channel protein.
 10. The immunogeniccomposition of claim 7, wherein replication of the virus in a subject isreduced relative to that of a virulent strain of the same virus, and thesubject is protected from challenge against a virulent strain of thesame species of virus.
 11. The immunogenic composition of claim 10,wherein the immune response to the peptide tag allows the subject to bedistinguished from a non-immunized subject.
 12. The immunogeniccomposition of claim 7, wherein the virus is influenza A selected fromthe group consisting of an H1N1 subtype, an H3N2 subtype, an H5N1subtype, and a combination thereof, the ion channel protein is M2, andthe peptide tag is at the carboxyl terminus of the protein.
 13. Theimmunogenic composition of claim 12, wherein the peptide tag is the mycantibody epitope tag, whose amino acid sequence consists of EQKLISEEDL(SEQ ID NO:1).
 14. The immunogenic composition of claim 13, wherein thecomposition comprises an attenuated influenza A virus and an attenuatedinfluenza B virus.
 15. A method for inducing a protective immuneresponse in a subject, the method comprising administering to thesubject an immunogenic composition comprising at least one liveattenuated RNA virus, the virus comprising an ion channel proteincomprising a peptide tag.
 16. The method of claim 15, wherein thepeptide tag is from about 3 amino acids to about 30 amino acids inlength.
 17. The method of claim 15, wherein the peptide tag is at thecarboxyl terminus of the ion channel protein.
 18. The method of claim15, wherein the replication and virulence of the virus in the subject isreduced relative to that of a virulent strain of the same virus, and thesubject is protected against challenge with a virulent strain of thesame species of virus.
 19. The method of claim 15, wherein the influenzavirus is a type A virus selected from the group consisting of an H1N1subtype, an H3N2 subtype, an H5N1 subtype, and a combination thereof,the ion channel protein is M2, and the peptide tag is at the carboxylterminus of the protein.
 20. The method of claim 19, wherein the peptidetag is the myc antibody epitope tag, whose amino acid sequence consistsof EQKLISEEDL (SEQ ID NO:1).